Assessment of Serum Magnesium Fractions in Workers Exposed to Pb from Pb-Battery Plant

Background: Most of studies assessed the effect of Pb-exposure on serum total magnesium (tMg). The hypomagnesium condition depended on protein concentration in the sample and influence of lifestyle factors. This study assessed the effect of Pb- exposure on serum tMg, corrected Mg (cMg), ionized Mg (iMg), percentage of iMg from tMg, and percentage of iMg from cMg with contemplation of lifestyle factors. Study design: Case control study. Methods: The serum magnesium fractions were assessed in 176 male Pb-exposed workers in the year 2015 at Tamil nadu in India and 80 control subjects with no occupational exposure of Pb. The serum tMg and albumin concentrations were estimated using diagnostic kit methods. Blood lead levels (BLLs) were estimated using atomic absorbtion spectrophotometer method. The fraction of cMg and iMg were calculated from serum tMg and albumin concentration among individual subjects. Results: The BLLs was significantly (P<0.001) increased in the study group as compared to control. Serum tMg, cMg, iMg, % of iMg from tMg and % of iMg from cMg concentrations were not significantly decreased in the study group as compared to control. Pb-exposure was significantly associated with abnormal frequency distribution of serum iMg (P=0.048) and % of iMg from tMg (P=0.016). Smoking habit was significantly associated with cMg (P=0.039) and % of iMg from cMg concentration (P=0.018). The alcohol consumption was significantly (P=0.049) associated with cMg. Conclusion: The Pb-exposure and lifestyle factor such as smoking and alcohol consumption were associated with alteration of serum magnesium fractions.

Introduction agnesium (Mg) is second most abundant intracellular cation in the body. The highest percentage of body Mg is found in bones followed by muscle and soft tissue 1 . It is a divalent cation and plays critical role in calcium and potassium transport, cell signaling, energy metabolism, gene stability, DNA repair, and replication 2 . Mg exists in blood in three forms such as ionized fraction (iMg), which comprises about 55% of serum tMg, the Mg bound to protein, particularly to albumin about 20% and Mg complexed to anions about 25%. These three fractions are in equilibrium with each other 3 .The serum fractions of tMg and iMg were closely related in hypermagnesaemia and poorly related in hypomagnesaemia, which depends on protein concentration 4 . The measurement of serum tMg overestimates the incidence of hypomagnesaemia when hypoalbuminemia is present 5 . Chronic alcoholics found reduced levels of serum tMg and iMg concentration than in the control 6 .The deficiency of serum iMg was associated with acute migraines 7 . Serum iMg, quotient Mg (iMg/tMg) and bound-Mg concentration were noted reduced levels in acute myocardial infarction patients and serum tMg was not altered 8 .Significantly decreased levels of iMg and bound-Mg (tMg-iMg) were reported in hyperthyroid patients 9 . About 3% increase of serum tMg concentration was reported in short-term occupational Pb-exposure 10 . The elevation of tMg caused by the increased release of Mg from the tissue due to its displacement from binding sites by Pb ions. Significantly decreased serum tMg and thiamine concentration was presented in Nigerian population exposed to Pb through their occupation and the low BLLs can enhance Pb absorption and also potentiate Pb-neurotoxicity in the presence of decreased serum tMg 11 . Significantly decreased of erythrocyte Mg was reported in workers, who had BLLs >20 µg/dL 12 . Moreover, decreased serum tMg was reported in occupational Pb-exposure 13 . In Pb-poisoning cases a significant drop of plasma Mg and without alteration of erythrocyte and urinary-Mg was reported 14 . Moreover, severe hypomagnesaemia was reported with persistent urinary loss of Mg in Pb-poisoning case 15 . Rats under subchronic Pb-intoxication were noted significantly reduced Mg in hard tissue and this reduction was due to competitive antagonism between Pb and Ca and Mg 16 . Rat intoxicated with ethanol plus Pb presented significantly decreased serum tMg, so it was concluded that the Pb-exposed human subject abusing alcohol might be vulnerable to accumulation of Pb in organs of the body and deficiency of bio-elements was associated with health injury 17 .

M
In some studies, hypomagnesium was also associated with oxidative stress, pro-inflammatory state, endothelial dysfunction, platelet aggregation, insulin resistance 18 , obesity 19 , acute exacerbation of COPD 20 , cardiovascular disease 21 , diabetes 22 , coronary artery disease 23 , lipid profiles 24 and smoking 25 . Mg deficiency was associated with reduction of serum sphingomyelin with elevations of lipid profiles (Cholesterol, LDL-C,VLDL-C and triglycerides) and oxidative stress, characterized by reduction in glutathione (GSH) and activation of e-NOS and n-NOS 26,27 .
Studies on short-term Pb-exposure showed an increased serum t-Mg. The chronic and Pb-poisoning case showed decreased concentration of serum tMg. The hypomagnesium condition was dependent on protein concentration 4 .Incidence of hypomagnesium overestimates the serum tMg measurement when hypoalbuminemia is present 5 . Serum tMg measurement does not reflect the biologically active Mg 2+ fraction. Serum iMg measurement provides better discrimination in normal and abnormal patients 28 . Decreased level of serum tMg was also associated with lifestyle factors such as obesity, smoking , alcohol consumption, hypertension and diabetes.
The present study has chosen the serum magnesium fractions such as tMg, iMg, cMg, % of iMg from tMg and % of iMg from cMg to assess the effect of Pb-exposure and lifestyle factors among workers from Pb-battery plant.

Methods
This case-control study, we enrolled 176 male Pb-battery manufacturing workers in the year 2015 at Tamil nadu in India and considered them as study group and 80 healthy subjects with no occupational exposure to Pb considered them as a control group. Serum Mg fractions were compared between study and controls.
The institutional Ethical Committee (IEC) approved the study with letter no.142/6/dated 3-12-2014. Subjects were informed about the study and consent was obtained before their participation in the study.
Using the mean difference of serum tMg reported in occupational Pb-exposure and controls the sample size was calculated 13 . Total sample size obtained for this study was198 with 119 study and 79 control samples. Sample size was calculated using openEpi info, version 3 with input data of confidence interval (CI) 85%, power 80%, allocation ratio 1.5 and difference between means is 0.1. The subjects with risk of cardiovascular disease, thyroid dysfunction, and diabetes were excluded.

Blood lead
The blood lead levels (BLLs) used as an indicator of Pbexposure. The BLLs was measured using the method of Barman et al 29 . In this method, two ml of whole blood sample was digested by a microwave digestion system (ETHOS-D, Italy) with 2 mL of nitric acid (HNO3) and 0.2 mL of hydrogen peroxide (H2O2). The digested samples were made up to 5 mL using triple distilled water and centrifuged. The BLL was measured by an atomic absorption spectrophotometer (GBC-Avanta, Australia). Twenty µg/dL of the standard solution were prepared from the lead standard solution and added to the lowest concentration of the sample. The analysis found 100% recovery with % of relative standard deviation at <0.5 for three replicates. The frequency distribution of BLLs among study and control groups were done by using OSHA standard 30 .

Body mass index and Blood pressure
BMI was calculated by using subjective weight (kg) and height (m) and expressed as kg/m 2 . Thefrequency distribution of BMI and blood pressure (SBP & DBP) among study and control group did by using WHO classification 31 and JNC 7 th report 32 respectively.

Serum magnesium
Serum total magnesium was determined using colorimetric and end point method. The diagnostic kit was manufactured by Linear Chemical SL, Joaquim Costa 18.2 planta, 08390 Montag, Barcelona, Spain. In this method, the specific binding of Calmagite, a metallochromic indicator and magnesium at alkaline pH with the resulting shift in the absorption wavelength at 520 nm. Intensity of the color formed is proportional to the concentration of Mg in the sample. Magnesium concentration in the samples was expressed as mg/dL. The detection limit of method is 0.01 mg/dL and linearity is up to 10 mg/dL. Corrected magnesium (cMg) and ionized magnesium (iMg) was calculated using serum tMg and albumin concentration with formulas .

Statistical analysis
All the data were analyzed SPSS version 20. The data was presented in mean and standard deviation and proportion. Independent t-test was used to find out the differences in age and serum Mg fractions between study and controls. Chisquare test was applied to show differences in BMI, SBP, DBP, BLLs, smoking, alcohol consumption and serum Mg fractions between study and controls. Spearman's correlation coefficient test was used to find out the association between BLLs and serum Mg fraction in study and control. The probability of less than 0.05 is considered as significant.

Serum albumin
The serum albumin concentration was measured by using Prietest clinical chemistry reagents 33 . This diagnostic kit was manufactured by Robnik (India) private limited, industrial area, Mahape, Navi Mumbai, India. In this approach, albumin in a buffered solution reacts with the anionic Bromocresol green dye and gives a green color measured at 628 nm. The intensity of green color was directly proportional to concentration of albumin present in the sample. The results were expressed as g/dL of sample.

Serum magnesium fraction
Percentage of iMg from tMg and percentage of iMg from cMg were calculated using the values off tMg, iMg and cMg.

Results
The characteristics of study and control groups are presented in Table 1.Variables of BMI, blood pressure (SBP & DBP), smoking and alcohol consumption of study group was suitable matched with control. BLLs in the study group were significantly (P<0.001) increased as compared to control. The levels of serum magnesium fraction such as serum tMg, cMg, iMg, % of iMg from tMg and % of iMg from cMg are reported in Table 2. The levels of serum Mg fractions (serum tMg, cMg, iMg, % of iMg from tMg and % of iMg from cMg) were not significantly decreased in the study group as compared to controls.
The normal and abnormal frequency distribution of serum Mg fraction was done using 5th percentile of control group. The abnormal frequency of serum iMg (P=0.048 is equal to one-tailed) and % of iMg from tMg (P=0.016 is equal to twotailed) levels were significantly decreased in the study group as compared to controls (Table 3).
Smoking habit significantly decreased the levels of cMg and % of iMg from cMg. Alcohol consumption was significantly decreased cMg.The other lifestyle factors such as BMI, SBP,and DBP among these subjects didnot influence the serum magnesium fraction (Table 4).  The serum magnesium fractions such as tMg, iMg cMg and % of iMg from tMg were negatively associated with BLLs in the study group. In the control group, the levels of tMg and iMg was negatively associated with BLLs and the levels of cMg, %of iMg from tMg and % of iMg from cMg was positively associated with BLLs (Table 5).

Discussion
The present study assessed the effects of Pb-exposure on serum magnesium fractions in workers exposed to Pb from Pbbattery plant. The measurement of BLLs was used as body burden of Pb-exposure. The Pb-exposure among study and control groups was assessed by using the OSHA regulation. 72.8% study group workers had BLLs < 40 µg/dL and 27.2% workers had BLLs >40µg/dL. In case of control group, 100% of workers had BLLs < 40 µg/dL. The magnesium deficiency was associated with hypertension, arrhythmia, arterial calcification, atherosclerosis, heart failure and an increased risk for thrombosis. The magnesium deficiency is a principal driver of cardiovascular disease and public health crisis 21 . A study, related to short-term Pb-exposure showed an increased serum tMg 10 . The chronic Pb-exposure and Pbpoisoning cases showed decreased serum tMg concentration [11][12][13][14][15] . During the present study, we noticed decreased serum tMg concentration in the study group as compared to control. The condition of hypomagnesium was dependent on protein concentration in sample 4 . The incidence of hypomagnesaemia overestimates through the measurement of serum tMg when hypoalbuminemia is reported 5 . The determination of serum tMg does not reflect the biologically active Mg 2+ fraction. A reduced level of serum tMg was related to lifestyle factors such as obesity 19 , smoking 22 , alcoholism 34 , high blood pressure 35 and diabetes 22 . The present study has chosen the serum magnesium fractions such as tMg, iMg, cMg, % of iMg from tMg and % of iMg from cMg to assess the effect of Pb-exposure and lifestyle factors among workers from Pb-battery plant. The parameters of iMg, cMg, % of iMg from tMg and % of iMg from cMg were obtained from serum tMg and serum albumin concentration. During the present study, we noted decreased levels of serum magnesium fractions (tMg, cMg, iMg, % of iMg from tMg and % of iMg from cMg) in the study group as compared to controls. Significantly altered abnormal frequencies of serum iMg and % of iMg from tMg levels were noted in the study group. The serum magnesium fractions such as tMg, iMg, cMg and % of iMg from tMg were negatively associated with BLLs in the study group. In control group, the levels of tMg and iMg was negatively associated with BLLs and the levels of cMg, % of iMg from tMg and % of iMg from cMg was positively associated with BLLs in controls.
Some studies reported decreased serum tMg concentration in smokers 21,36 . Niemela et al 37 reported decreased serum iMg in smokers. During the present study, we assessed the influence of smoking habit on serum Mg fractions and found significantly decreased levels of cMg and % of iMg from cMg. Among smokers, the levels of serum tMg,iMg and % of iMg from tMg were not altered. Smoking does not affect the serum mineral levels including magnesium 38 . Significantly reduced serum tMg and iMg concentration was reported in chronic alcoholics 6 . In the present study, alcohol consumption was noted significantly decreased in cMg levels. The alcohol consumption pattern among these subjects does not influence serum magnesium fraction because these subjects were not chronic alcoholics.

Conclusion
The Pb-exposure and lifestyle factors such as smoking and alcohol consumption were associated with alteration of serum magnesium fractions.BMI, DBP, and SBP did not influence serum Mg fractions. 12. Chiba M, Shinohara A, Matsushita K, Watanabe H, Inaba Y. Indices of lead-exposure in blood and urine of lead-exposed workers and concentrations of major and trace elements and activities of SOD, GSH-Px and catalase in their blood. Tohoku J Exp Med. 1996; 178(1): 49-62.